Introduction. Effective precise knock-in is crucial for implementing CRISPR-Cas9 system as an efficient instrument for potential gene therapy. Homology directed repair (HDR) pathway allows correction of all types of existing mutations. However, HDR is not a major repair pathway of the cell that limits its efficiency. In our study, we present for the first time how repair factors NUDT16L1 controls HDR efficiency. Aim: to study an influence of NUDT16L1 knockdown and overexpression on the HDR efficacy. Methods. HEK293T culture was used to perform the research. Plasmid CRISPR-Cas system along with NUDT16L1 overexpression vector were delivered with lipofection. For NUDT16L1 knockdown small interfering RNAs were used. Results. We discovered that knockdown of NUDT16L1 enhances HDR both in the plasmid and genomic loci increasing eGFP signal from 1.8 to 3.6 times in HEK293T cells. Conclusion. NUDT16L1 knockdown could be used for enhancing of the pathogenic mutations correction through genome editing.

Background. Retinoblastoma is a childhood malignant tumor caused by biallelic inactivation of the RB1 gene. Early molecular genetic diagnosis of retinoblastoma is necessary both for an adequate choice of an algorithm for treating a patient, and for competent medical genetic counseling of the family Objective. To establish the frequency and spectrum of mutations in the RB1 gene in the group of patients with retinoblastoma. Methods. The study was carried out on the DNA of blood lymphocytes from 492 patients with retinoblastoma. Screening of point mutations, small insertions/deletions in the RB1 gene was performed by semiconductor high-throughput parallel sequencing. Exclusion of gross deletions in the RB1 gene was performed by MLPA. Results. 492 unrelated patients with retinoblastoma were studied, including 38.2% (188/492) with bilateral form and 61.8% (304/492) with unilateral form. In the group of patients with bilateral retinoblastoma, germline mutation was found in 96.8% (182/188) patients, and in the group of unilateral patients, in 16.4% (50/304). In total, the RB1 gene in the studied group of patients 339 mutations were found, 232 germline and 107 somatic. An almost complete spectrum of molecular changes was revealed, including nonsense mutations, 37.5% (127/339); missense mutations, 5.3% (18/339); frame shift mutations, 18.9% (64 / 339); splice site mutations, 13.9% (47/339); and large deletions, 24.5% (83/339). Conclusion. The use of deep high-throughput parallel sequencing and the MLPA method allows efficient detection of molecular genetic changes in the RB1 gene. The types of mutations found in the studied group, their frequency and distribution are the same as the results of researchers in other countries.

Background. The identification and comprehensive characterization of new molecular markers of malignant tumors remains an urgent task of oncology. Earlier, according to the results of a genome-wide screening of differential DNA methylation of normal and tumor breast tissues, abnormal demethylation of the CpG island of LTB4R/LTB4R2 genes in tumors relative to norm was revealed. Aim. The present study focuses on the molecular and clinical characterization of abnormal demethylation of this CpG island and expression of the LTB4R/LTB4R2 genes in breast cancer (BC). Methods. For exploratory analysis, we used the previously obtained results of genome wide bisulfite sequencing of breast cancer (BC) and normal mammary gland samples. From this set, 10 samples of triple-negative (TN) breast cancer with abnormal LTB4R/LTB4R2 demethylation and 6 normal breast samples were selected, for which a comparative analysis of CpG methylation levels in cancer vs norm using the Mann-Whitney test was performed. Sanger bisulfite sequencing was used to confirm abnormal demethylation in TN BC samples. The analysis of the obtained electrophoregrams was carried out using the SeqBase software developed by the authors. To validate and assess the level of predictive significance of LTB4R/LTB4R2 gene expression, level 3 data were used to measure methylation from Illumina HumanMethylation 450K arrays, RNA-seq expression, as well as clinical characteristics for 731 samples from the TCGA-BRCA project. Kaplan-Meier curves were compared using a logrank test. Results. Abnormal demethylation of LTB4R/LTB4R2 genes in breast tumors was confirmed by Sanger sequencing. Among samples from the TCGA-BRCA project, a group of TN BC samples with low methylation of LTB4R/LTB4R2 genes was identified. Overall survival was significantly reduced in the TN breast cancer group with high LTB4R expression, as well as in the group of normal-like breast tumors with low LTB4R expression, and in the LumB group overall survival was significantly reduced when tumors demonstrated low LTB4R2 expression. Conclusions. Fine mapping of abnormal demethylation in individual CpG dinucleotides of the LTB4R/LTB4R2 genes will make it possible to design an effective PCR system that may potentially be used to define patients with TN breast cancer that would benefit from leukotriene receptor therapeutic inhibition. Different expression levels of LTB4R/LTB4R2 allow their use as a prognostic marker for breast cancer, but the prognosis directly depends on the molecular subtype of a tumor. This also applies to the use of different levels of LTB4R/LTB4R2 expression as a predictive marker of the sensitivity of such tumors to leukotriene receptor inhibitors in case they enter clinical trials.

The rs13128867 (T>C) polymorphism located between SLC7A11 and PCDH18 genes was originally described as a new susceptibility locus for Kawasaki disease (KD) in the Chinese population. Focusing on the identification of risk alleles for juvenile-onset autoimmune rheumatic diseases, the presented paper aims to demonstrate the results of the study undertaken to investigate an association between the rs13128867 polymorphism and KD, juvenile idiopathic arthritis (JIA), and juvenile systemic lupus erythematosus (JSLE) in Caucasians living in Belarus. In total, 675 children and adolescents under 17 at the onset of the disease were included in the study performed, and 386 out of them were without any autoimmune, inflammatory, or joint disorders and served as the clinical control. Genomic DNA samples were genotyped using the real-time PCR technique. Case-control comparison was adjusted for sex with a view of reducing uneven gender distribution between the groups, and p-values were adjusted with the Benjamini-Hochberg procedure. The rs13128867 minor C allele frequency in the control group was found to be 17.6% that is close to other European populations. At the same time, an associative analysis of the rs13128867 polymorphism with KD did not replicate the data known for the Chinese population. Further analysis revealed an association between the rs13128867 minor allele with JIA (OR = 1.50, 95% CI 1.11-2.03, padj = 0.027) and oligoarthritis - the most common JIA subtype (OR = 1.67, 95% CI 1.17-2.39, padj = 0.016). A tendency to association of the rs13128867 minor allele and JSLE was also observed. Thus, the study results demonstrated for the first time that the SLC7A11-PCHD18 rs13128867 polymorphism may be a novel genetic risk factor for JIA in Caucasian patients. However, a further investigation into the rs13128867 polymorphism is strongly required.

A common pathogenic link in type 2 diabetes mellitus (T2D) and coronary artery disease (CAD) is oxidative stress, which develops as a result of an imbalance in the production of reactive oxygen species (ROS) and their neutralization by the antioxidant defense system. Neutrophilic cytosolic factor 4 (NCF4) is directly involved in the synthesis of superoxide anion as part of NADPH oxidase. In this regard, the purpose of this study was to investigate the associations of eight single nucleotide polymorphisms of the NCF4 gene rs5995355 (A>G), rs5995357 (T>A), rs1883112 (G>A), rs4821544 (G>A), rs760519 (T>C), rs729749 (C>T), rs2075938 (G>A), rs2075939 (C>T) with a predisposition to T2D, as well as the risk of developing CAD in patients with T2D. The study included 1579 patients with T2D (448 of them were also diagnosed with CAD) and 1627 relatively healthy volunteers. Genotyping was performed using MALDI-TOF mass spectrometry on the MassArray Analyzer 4 platform. Statistical processing of the obtained data was carried out using the SNPStats online program. The allele and genotype frequencies of the studied SNPs in T2D patients did not differ from those in the control group (p>0.05). Associations of genotypes rs4821544-C/C (OR 1.71, 95CI 1.12-2.59, p=0.013) and rs5995357-A/A (OR 3.74, 95CI 1.14-12.31, p=0.026) with a predisposition to CAD in diabetic females were established. Despite the absence of associations of the studied SNPs NCF4 with CAD in males, associations of the haplotype structure of NCF4 (p=0.0064), as well as the haplotypes H2 (OR 1.79, 95CI 1.16-2.76, p=0.0085) and H3 (OR 1.77, 95CI 1.06-2.97, p=0.03) with an increased risk of CAD were observed exclusively in diabetic males. In addition, a sex-independent relationship of the rs4821544-C/C genotype with an increased level of glycated hemoglobin (p=0.032) and oxidized glutathione (p=0.049) was revealed in patients with CAD and T2D. In the same category of patients haplotypes H4 rs5995355G-rs5995357A-rs1883112G-rs4821544C-rs760519T-rs729749C-rs2075938G-rs2075939C and H10 rs5995355A-rs5995357T-rs1883112G-rs4821544C-rs760519T-rs729749C-rs2075938A-rs2075939C of NCF4 gene were associated with an increase in the content of HbA1c 8.67 % (p=0.011) and 6.27% (p=0.038), respectively. The data obtained indicate a significant contribution of the NCF4 gene polymorphism to the pathogenesis of CAD in patients with T2D and create a scientific basis for the development of targeted therapy and prevention of this pathology.

A number of diseases, including cancer, are associated with accumulation of GC-enriched sequences in the general cell-free DNA (cfDNA) pool. This fact is primarily explained by these DNA fragments increased resistance to the blood nucleases which can activate DNA sensors - TLR9 and AIM2 that have a different effect on the cancer cells functioning. The aim of the present study was to study the MCF 7 cells biological response to GC-rich cfDNA sequences and using AIM2 and TLR9 knockout to identify the functional role of DNA-binding receptors in the development of cellular adaptive response. It was experimentally established that MCF7 culture cells with TLR9 and AIM2 receptors “turned off” respond to stimulation by GC-DNA fragments by reducing the transcriptional activity of the genes of the TLR9/MYD88/NF-KBsignaling pathway and associated STAT 3/6 signaling pathways that increase the survival of cancer cells. This shows the need for further studies of the role of other genes, including on the example of cell lines with TLR9 and AIM2 knockout, in terms of detailing the mechanisms of the effects noted in this work on cancer cell survival.